Process for the production of alcoholic beverages using maltseed

ABSTRACT

The invention relates to a process for the production of alcoholic beverages such as beer or whiskey with a mixture of enzymes comprising an endo-β(1,4)-xylanase, an arabinofuranosidase, an alpha-amylase, an endo-protease and a β-(1,3; 1,4)-glucanase, and optionally, a saccharifying amylase and/or an exopeptidase. Preferable are mixtures in which the enzymes which are necessary in the brewing process are provided by transgenic seeds. Only a minor amount of malt may be necessary for flavour and color.

FIELD OF THE INVENTION

[0001] The present invention relates to a process for the production ofalcoholic beverages, especially beer and whiskey.

BACKGROUND OF THE INVENTION

[0002] Alcoholic beverages such as beer can be manufactured from maltedand/or unmalted barley grains. Malt, in addition to yeast, contributesto flavour and color of the beer. Furthermore, malt functions as asource of fermentable sugar and enzymes. Whether malt is used in thebrewing process depends on the type of beer and on the country where thebeer is produced. In African countries, for example, there is notradition of using malt.

[0003] The general process of malting and brewing is recently describedby R. C. Hoseney (Cereal Foods World, 39(9), 675-679, 1994). Malting isthe process of controlled germination followed by controlled drying ofthe barley grain. Grain is converted into malt by successive steps ofsteeping, germination, growth and drying (kilning). In this respect, thegermination step is important to obtain expression of a series ofenzymes which enables modification of the endosperm. This modificationproduces fermentable carbohydrates.

[0004] The subsequent drying/heating step of the malting processproduces flavour and color due to non-enzymatic browning (Maillard)reactions.

[0005] The process of malting is a very complicated and costly part ofthe beer production process. Several disadvantages of the maltingprocess can be mentioned:

[0006] the enzyme level of malt is variable which leads to unpredictableresults,

[0007] not every enzyme activity which is desirable is formed or isformed in sufficient amounts during germination, which makes enzymesupplementation necessary,

[0008] conditions which favour high flavour and color may be deleteriousfor enzyme activity of the malt,

[0009] the process is expensive,

[0010] 10- 20% loss in weight occurs during germination, due torespiration and growth of rootlets (which are removed during cleaning ofthe malt),

[0011] it is not possible to produce malt at any place which isdesirable, because of unfavourable climate conditions,

[0012] the use of malt can lead to colloidal instability because ofsolubilization of protein by protease present in the malt,

[0013] formation of biogenic amines can occur (J. Food Science 59(5),1104-1107, 1994), which may lead to e.g. histaminic intoxication.

[0014] Traditionally, malt was the only source of fermentablecarbohydrates and enzymes, and in many countries it still is. However,to date more and more beers are produced using other sources ofcarbohydrates than malt and/or barley, i.e. virtually any starch sourceor liquefied/degraded starch, the so-called adjuncts. Since malt notonly functions as a source of fermentable carbohydrate, but also as asource of enzymes, alternative enzyme sources have to be provided uponsubstitution of more than approximately 50% malt with unmalted barleyand/or with adjuncts. Moreover, malt gives the beer flavour and color.

[0015] In the production of malt there is a trade-off between flavourand color and enzyme activity. A malt providing high flavour and darkcolor can only be produced after more extensive drying at relativelyhigh temperatures. These are conditions which are deleterious for theactivity of an enzyme. Thus the supplementation of enzymes from anexogenous source is necessary from several points of view. In thatrespect, the use of microbial enzymes has been common practice for sometime. For example, for brewing beer grains and/or malted grains areliquefied and saccharified in order to yield fermentable sugars.Liquefaction steps may be improved by the use of thermostablealpha-amylases as described in for instance U.S. Pat. No. 4,285,975 orU.S. Pat. No. 5,180,669. Also proteases are used to increase the amountof freely available nitrogen in the wort to improve fermentation.

[0016] Apart from starch other polysaccharides are present in cerealgrains as for example β-glucans (Henry, R. J. et al. J. Sci. Food Agric.36, 1243-1253, 1985). The β-glucanases present in malt are notsufficiently thermostable to be active during the brewing process. Theseβ-glucans are highly viscous and give wort and beer filtration problems.This is the reason why microbial β-glucanases are widely used in thebrewing process.

[0017] Non-starch polysaccharides also include pentosans, the structureof which has been widely studied recently (Gruppen, H. et al. Carbohydr.Res. 233, 45-64, 1992), in particular those of barley and malt (Vietor,R. J. et al. Carbohydr. Res. 254, 245-255, 1994). A pentosanase fromPenicillium emersonii has been said to improve the production andextraction of fermentable sugars in brewing (GB 2,150,933).

[0018] The use of xylanase B to improve wort quality has also beenmentioned in WO94/14965.

[0019] Despite the advance which has been made in this area, there isstill a need for methods of beer brewing with enzyme preparations foruse therein.

SUMMARY OF THE INVENTION

[0020] The present invention discloses a process for the production ofalcoholic beverages, such as beer, to which a mixture of enzymes isadded, which mixture comprises at least an endo-β(1,4)-xylanase, anarabinofuranosidase, an alpha-amylase, an endo-protease and aβ-(1,3;1,4)-glucanase, optionally also containing a saccharifyingamylase and/or an exo-peptidase. Preferably the enzymes that arenecessary for the beer production process are provided by transgenicseeds.

[0021] The present invention further discloses transgenic seedsexpressing the enzymes necessary in the beer production process.

DESCRIPTION OF THE INVENTION

[0022] We have now surprisingly found that the brewing process can beperformed in the presence of the mixture of enzymes as claimed, with aminimal amount of malt. This process has been performed to manufacture aclassical malt beer, but it can equally well be performed in any processwhere malt is used to provide enzyme activities.

[0023] The enzymes to be used are selected from the group of enzymeswhich are necessary in the brewing process. They include enzymes whichare selected from the group of amylolytic enzymes, from the group ofcellulolytic enzymes, from the group of hemicellulolytic enzymes andfrom the group of proteolytic enzymes.

[0024] Amylolytic enzymes comprise enzymes like alpha-amylase,saccharifying amylase, amyloglucosidase, exo-amylase, pullulanase.

[0025] Cellulolytic enzymes comprise enzymes like β-1,4-endoglucanase,cellobiohydrolase, β-glucosidase.

[0026] Hemicellulolytic enzymes comprise enzymes likeβ-1,3-1,4-glucanase, xylanase, endo-arabinanase, arabinofuranosidase,arabinoxylanase, arabinogalactanase, ferulic acid esterase.

[0027] Proteolytic enzymes comprise enzymes like exopeptidases andendopeptidases (also called prote(in)ases).

[0028] In this respect, the choice for a specific amylolytic,cellulolytic, hemicellulolytic or proteolytic enzyme is not critical forthe present invention, besides that the choice of the enzyme should besuch that the properties of the enzyme (such as pH and temperaturerange) are compatible with the specific circumstances in the brewingprocess.

[0029] Numerous genes encoding amylolytic, cellulolytic,hemicellulolytic and proteolytic enzymes are available to the skilledperson. The genes encoding the enzymes of interest can be obtainablefrom any source, plant, animal or microbial. Preferably, the genes areobtainable from a microbial source.

[0030] The endo-β-1,4)-xylanase can be obtained from a culture ofAspergillus niger (EP 0 463 706). Arabinofuranosidase is available asisoenzyme A or isoenzyme B (EP 0 506 190) or arabinoxylanhydrolase (EP 0730 653) from a culture of Aspergillus niger. The thermostable amylaseis derivable from Bacillus licheniformis (WO91/14772, WO92/05259), andis, for example, commercially available under the tradename BrewersAmyliq Thermostable (B.A.T.S.). Activity of the thermostable amylase isexpressed in TAU units. The endoprotease can be derived from Bacillusamyloliquefaciens, and is also commercially available under thetradename Brewers protease (+) and its activity is expressed in PCunits. From the same bacterium also the β-(1,3;1,4)-glucanase can bederived (Hofemeister et al., Gene 49, 177, 1986), which is alsocommercially available under the tradename Filtrase L 3000 (+). Activityof the glucanased is expressed in BGLU units. The optional saccharifyingamylase can also be obtained from commercially available sources(amylase from Aspergillus oryzae under the tradename Brewers Fermex forwhich the activity is expressed in FAU units), but it can also beobtained from a pure culture of Penicillium emersonii (available at theATCC (American Type Culture Collection) under number ATCC16479). Theoptional exopeptidase can be derived from a pure culture of Aspergillussojae, as has been deposited at the Centraal Bureau for Schimmelcultures(CBS), Oosterstraat 1, Baarn, The Netherlands, under number CBS 209.96(A. sojae (DS 8351) at Feb. 12, 1996).

[0031] The enzyme activities which are currently known to be necessaryfor beer production are alpha- and β-amylase to convert the starch ofthe endosperm to fermentable sugars, protease to degrade protein intosoluble nitrogen compounds which function as yeast nutrients, andβ-glucanase and xylanase to hydrolyse barley β-1,3-1,4-glucans andxylans, respectively, to oligosaccharides which results in a reductionof viscosity and an improvement of filterability. Thus, to provide allnecessary enzymes from an exogenous source, i.e. a microbial source,addition of at least 5 different enzymes would be required. Although onemicro-organism may be used for the production of all enzymes, differentfermentation conditions will be required for optimal production of allenzymes.

[0032] By using enzymes from microbial sources, some of thedisadvantages of the malting process which have been mentioned before,can be overcome. However, the use of microorganisms as a source ofenzymes also has its disadvantages:

[0033] a series of different fermentations of at least one microorganismis required to obtain each enzyme in a sufficient amount,

[0034] the enzyme preparations may contain undesirable side activities,

[0035] consumers do not favour additions other than plant material,water and yeast,

[0036] limited stability of an enzyme preparation during storage at roomtemperature.

[0037] The general use of transgenic seeds containing an enhanced amountof enzyme in industrial enzyme-catalyzed processes is described ininternational patent application WO91/14772. The direct use of theenzyme-containing seed in an industrial-process circumvents the need forfirst extracting and/or isolating the enzyme. The seed can function as astable and manageable storage form of enzymes.

[0038] For one single enzyme, β-1,3-1,4-glucanase, the expression inbarley seeds has been mentioned as an alternative for exogenousaddition.

[0039] East-German patent application DD 275704 discloses theconstruction of an expression vector to enable the seed-specificexpression of a Bacillus β-1,3-1,4-glucanase in barley. However, at thattime effective transformation of barley was not yet known. Using theseeds expressing the Bacillus β-glucanase, a higher amount of grain cansubstitute for malt without obtaining serious filtration problems.However, other enzyme activities which are necessary in the beer brewingprocess still have to be obtained from malt or supplemented exogenously.

[0040] Mannonen et al. (1993) suggest the incorporation of a fungalβ-1,3-1,4-glucanase in barley seeds. In this way, the brewing processwould be improved by the expression in the seed of a β-1,3-1,4-glucanasewhich has a higher thermostability than the endogenous barley enzyme. Inthis case, however, the intention is to take the seed through the normalmalting process. Moreover, also in this case other enzyme activitieswhich are necessary in the beer brewing process still have to beobtained from malt or supplemented exogenously.

[0041] In a preferred process of the present invention, the enzymeswhich are necessary in the brewing process are expressed in transgenicseeds. The thus-produced transgenic seeds expressing said necessaryenzymes are in turn used in the beer brewing process. In this way, theuse of malt is reduced, whereas the addition of exogenous microbialenzymes is circumvented.

[0042] The transgenic seeds expressing the enzymes which are necessaryin the beer brewing process are covered by the general name MaltSeed.

[0043] Plant genera which are capable of producing the enzyme ofinterest in their seeds include the species of which the grains orproducts from grains have a history of use in beer brewing. However,also plant species which are not commonly used for beer brewing may beused as a source of transgenic seed expressing an enzyme of interest,especially in those cases that only a minor amount of said transgenicseed is added to the brewing process. Plant genera which qualify thesecriteria are, for instance, barley, corn, rice, wheat, sorghum, millet,oats, cassava and the like.

[0044] The gene encoding an enzyme of interest is expressed in the plantseed using regulatory sequences functional in a plant or plant seed.Those regulatory sequences include promoter sequences, terminatorsequences and, optionally, transcription enhancer sequences.

[0045] Promoter sequences may be used which lead to constitutiveexpression of the gene in the whole plant. Otherwise, promoter sequencesmay be used which are active in directing expression of the gene to theplant seed.

[0046] Furthermore, the expression of an enzyme of interest can bedirected either to a specific cellular compartment, such as cytosol,endoplasmatic reticulum, vacuole, protein body, or to the extracellularspace, using specific targeting sequences.

[0047] The choice for a specific cellular compartment or for theextracellular space depends on the properties of the enzyme of interestand should be made in such a way that an optimal environment for theenzyme is created. For instance, the enzyme of interest should be in anenvironment that allows optimal stability of the protein during seedmaturation. In addition, the enzyme of interest should be in anenvironment where expression of the enzyme does not inhibit essentialplant metabolic processes or lead to a deleterious effect on the plantor seed viability.

[0048] In order to be capable of being expressed in a plant cell a DNAsequence coding for the enzyme of choice will usually be provided withregulatory elements enabling it to be recognised by the biochemicalmachinery of the host and allowing for the open reading frame to betranscribed and translated in the host. It will usually comprise atranscriptional initiation region which may be suitably derived from anygene capable of being expressed in the plant cell, as well as atranslational initiation region for ribosome recognition and attachment.In eukaryotic plant cells, such an expression cassette usually comprisesin addition a transcriptional termination region located downstream ofsaid open reading frame, allowing transcription to terminate andpolyadenylation of the primary transcript to occur. In addition, thecodon usage may be adapted to accepted codon usage of the host plant ofchoice. The principles governing the expression of a DNA construct in aplant cell are commonly understood by those of ordinary skill in the artand the construction of expressible chimeric DNA constructs is nowroutine.

[0049] A special type of replicon is one capable of transferring itself,or a part thereof, to another host cell, such as a plant cell, therebyco-transferring the open reading frame coding for the enzyme(s)according to the invention to said plant cell. Replicons with suchcapability are herein referred to as vectors. An example of such avector is a Ti-plasmid vector which, when present in a suitable host,such as Agrobacterium tumefaciens, is capable of transferring part ofitself, the so-called T-region, to a plant cell. Different types ofTi-plasmid vectors (vide: EP 0 116 718 B1) are now routinely being usedto transfer chimeric DNA sequences into plant cells, or protoplasts,from which new plants may be generated which stably incorporate saidchimeric DNA in their genomes. A particularly preferred form ofTi-plasmid vectors are the so-called binary vectors as claimed in (EP 0120 516 B1 and U.S. Pat. No. 4,940,838). Other suitable vectors, whichmay be used to introduce DNA according to the invention into a planthost, may be selected from the viral vectors, e.g. non-integrative plantviral vectors, such as derivable from the double stranded plant viruses(e.g. CaMV) and single stranded viruses, gemini viruses and the like.The use of such vectors may be advantageous, particularly when it isdifficult to stably transform the plant host as is for instance the casewith woody species, especially trees and vines.

[0050] The expression “host cells incorporating a chimeric DNA sequenceaccording to the invention in their genome” shall mean to comprisecells, as well as multicellular organisms comprising such cells, oressentially consisting of such cells, which stably incorporate saidchimeric DNA into their genome thereby maintaining the chimeric DNA, andpreferably transmitting a copy of such chimeric DNA to progeny cells, beit through mitosis or meiosis. According to a preferred embodiment ofthe invention plants are provided, which essentially consist of cellswhich incorporate one or more copies of said chimeric DNA into theirgenome, and which are capable of transmitting a copy or copies to theirprogeny, preferably in a Mendelian fashion. By virtue of thetranscription and translation of the chimeric DNA according to theinvention those cells will produce the enzymes. Although the principleswhich govern transcription of DNA in plant cells are not alwaysunderstood, the creation of chimeric DNA capable of being expressed isnow routine. Transcription initiation regions routinely in use forexpression of the transformed polynucleotide in a constitutive way arepromoters obtainable from the cauliflower mosaic virus, notably the 35SRNA and 19S RNA transcript promoters and the so-called T-DNA promotersof Agrobacterium tumefaciens. In particular to be mentioned are thenopaline synthase promoter, octopine synthase promoter (as disclosed inEP 0 122 791 B1) and the mannopine synthase promoter. In addition plantpromoters may be used, which may be substantially constitutive, such asthe rice actin gene promoter. For seed-specific expression, a gene ofcDNA of interest can be inserted behind promoters originating from geneswhich are specifically expressed in the plant seed. These promotersinclude promoters of genes encoding seed storage proteins, such as theBrassica napus cruciferin promoter, the Phaseolus vulgaris phaseolinpromoter, the glutelin promoter from Oryza sativa, the zein promoter ofZea mays and the hordein promoter of Hordeum vulgare.

[0051] It is further known that duplication of certain elements,so-called enhancers, may considerably enhance the expression level ofthe DNA under its regime (vide for instance: Kay R. et al. (1987),Science 236, 1299-1302: the duplication of the sequence between −343 and−90 of the CaMV 35S promoter increases the activity of that promoter).In addition to the constitutive 35S promoter, singly or doubly enhanced,examples of high-level promoters are the light-inducible ribulosebisphosphate carboxylase small subunit (rbcSSU) promoter and thechlorophyll a/b binding protein (Cab) promoter. Also envisaged by thepresent invention are hybrid promoters, which comprise elements ofdifferent promoter regions physically linked. A well known examplethereof is the so-called CaMV enhanced mannopine synthase promoter (U.S.Pat. No. 5,106,739), which comprises elements of the mannopine synthasepromoter linked to the CaMV enhancer.

[0052] Specifically with monocot transformation the use of intronsbetween promoter and selectable marker gene enhances expression. Theterm “promoter” thus refers to a region of DNA upstream from thestructural gene and involved in recognition and binding RNA polymeraseand other proteins to initiate transcription. A “plant promoter” is apromoter capable of initiating transcription in plant cells. A“constitutive promoter” is a promoter which is active under mostenvironmental conditions and states of development or celldifferentiation.

[0053] As regards the necessity of a transcriptional terminator region,it is generally believed that such a region enhances the reliability aswell as the efficiency of transcription in plant cells. Use thereof istherefore strongly preferred in the context of the present invention.

[0054] Although some of the embodiments of the invention may not bepracticable at present, e.g. because some plant species are as yetrecalcitrant to genetic transformation, the practicing of the inventionin such plant species is merely a matter of time and not a matter ofprinciple, because the amenability to genetic transformation as such isof no relevance to the underlying embodiment of the invention.

[0055] Transformation of plant species is now routine for an impressivenumber of plant species, including both the Dicotyledoneae as well asthe Monocotyledoneae. In principle any transformation method may be usedto introduce chimeric DNA according to the invention into a suitableancestor cell. Methods may suitably be selected from thecalcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,1982, Nature 296, 72-74; Negrutiu I. et al, June 1987, Plant Mol. Biol.8, 363-373), electroporation of protoplasts (Shillito R. D. et al., 1985Bio/Technol. 3, 1099-1102), microinjection into plant material (CrosswayA. et al., 1986, Mol: Gen. Genet. 202, 179-185), (DNA or RNA-coated)particle bombardment of various plant material (Klein T. M. et al. 1987,Nature 32, 70), infection with (non-integrative) viruses, in plantaAgrobacterium tumefaciens mediated gene transfer by infiltration ofadult plants or transformation of mature pollen or microspores (EP 0 301316) and the like. A preferred method according to the inventioncomprises Agrobacterium-mediated DNA transfer. Especially preferred isthe use of the so-called binary vector technology as disclosed in EP A120 516 and U.S. Pat. No. 4,940,838).

[0056] Although considered somewhat more recalcitrant towards genetictransformation, monocotyledonous plants are amenable to transformationand fertile transgenic plants can be regenerated from transformed cellsor embryos, or other plant material. Presently, preferred methods fortransformation of monocots are microprojectile bombardment of embryos,explants or suspension cells, and direct DNA uptake or (tissue)electroporation (Shimamoto, et al, 1989, Nature 338, 274-276).Transgenic maize plants have been obtained by introducing theStreptomyces hygroscopicus bar-gene, which encodes phosphinothricinacetyltransferase (an enzyme which inactivates the herbicidephosphinothricin), into embryogenic cells of a maize suspension cultureby microprojectile bombardment (Gordon-Kamm, 1990, Plant Cell, 2,603-618). The introduction of genetic material into aleurone protoplastsof other monocot crops such as wheat and barley has been reported (Lee,1989, Plant Mol. Biol. 13, 21-30). Wheat plants have been regeneratedfrom embryogenic suspension culture by selecting embryogenic callus forthe establishment of the embryogenic suspension cultures (Vasil, 1990Bio/Technol. 8, 429-434). The combination with transformation systemsfor these crops enables the application of the present invention tomonocots.

[0057] Monocotyledonous plants, including commercially important cropssuch as rice and corn are also amenable to DNA transfer by Agrobacteriumstrains (vide WO 94/00977; EP 0 159 418 B1; Gould J, Michael D, HasegawaO, Ulian E C, Peterson G, Smith R H, (1991) Plant. Physiol. 95,426-434).

[0058] For barley a preferred transformation method has been describedin Tingay, S. et al. (The Plant J. 1(6),. 1369-1376, 1997).

[0059] To obtain transgenic plants capable of constitutively expressingmore than one chimeric gene, a number of alternatives are availableincluding the following:

[0060] A. The use of DNA, e.g. a T-DNA on a binary plasmid, with anumber of modified genes physically coupled to a second selectablemarker gene. The advantage of this method is that the chimeric genes arephysically coupled and therefore migrate as a single Mendelian locus.

[0061] B. Cross-pollination of transgenic plants each already capable ofexpressing one or more chimeric genes, preferably coupled to aselectable marker gene, with pollen from a transgenic plant whichcontains one or more chimeric genes coupled to another selectablemarker. Afterwards the seed, which is obtained by this crossing, maybeselected on the basis of the presence of the two selectable markers, oron the basis of the presence of the chimeric genes themselves. Theplants obtained from the selected seeds can afterwards be used forfurther crossing. In principle the chimeric genes are not on a singlelocus and the genes may therefore segregate as independent loci.

[0062] C. The use of a number of a plurality chimeric DNA molecules,e.g. plasmids, each having one or more chimeric genes and a selectablemarker. If the frequency of co-transformation is high, then selection onthe basis of only one marker is sufficient. In other cases, theselection on the basis of more than one marker is preferred.

[0063] D. Consecutive transformation of transgenic plants alreadycontaining a first, second, (etc.), chimeric gene with new chimeric DNA,optionally comprising a selectable marker gene. As in method B, thechimeric genes are in principle not on a single locus and the chimericgenes may therefore segregate as independent loci.

[0064] E. Combinations of the above mentioned strategies.

[0065] The actual strategy may depend on several considerations such asthe purpose of the parental lines (direct growing, use in a breedingprogramme, use to produce hybrids) but is not critical with respect tothe described invention.

[0066] It is known that practically all plants can be regenerated fromcultured cells or tissues. The means for regeneration vary from speciesto species of plants, but generally a suspension of transformedprotoplasts or a petri plate containing transformed explants is firstprovided. Shoots may be induced directly, or indirectly from callus viaorganogenesis or embryogenesis and subsequently rooted. Next to theselectable marker, the culture media will generally contain variousamino acids and hormones, such as auxins and cytokinins. It is alsoadvantageous to add glutamic acid and proline to the medium. Efficientregeneration will depend on the medium, on the genotype and on thehistory of the culture. If these three variables are controlledregeneration is usually reproducable and repeatable.

[0067] After stable incorporation of the transformed gene sequences intothe transgenic plants, the traits conferred by them can be transferredto other plants by sexual crossing. Any of a number of standard breedingtechniques can be used, depending upon the species to be crossed.

[0068] In one embodiment of the present invention, transgenic seeds areused which are engineered in such a way that these produce oneindividual enzyme, allowing for the flexible production of enzymemixtures with every enzyme activity ratio which is desired. In anotherembodiment, more than one enzyme activity may be contained in the seedsof an individual transgenic plant line.

[0069] The transgenic seeds containing the enzymes of interest can beadded together at a desired stage of the brewing process.

[0070] Alternatively, transgenic seeds containing an enzyme of interestcan be added individually, each at a desired point of the process.

[0071] The process of the present invention enables the development of amalt with a high level of flavour and color, without having to deal withthe enzyme activity of the malt. In the process of the present inventionthe use of malt is largely bypassed. Only a minor amount of malt maystill be necessary to provide the beer with flavour and color.

[0072] The transgenic seeds containing the desired enzymes can beapplied in the most optimal ratio with the grain and, optionally, with asufficient amount of malt for flavour and color. The transgenic seedscan be mixed beforehand with the grain and the malt. Alternatively, eachcompound, grain, transgenic seed and malt, can be added at separatestages of the beer brewing process.

[0073] Preferably, the individual compounds, transgenic seeds, grain andmalt, or the mixture of transgenic seed, grain and malt are milledbefore addition to the brewing process.

[0074] The transgenic seed contains the desired enzymes at an averagelevel that ranges from 0.001-2.5%, preferably from 0.01-1.0%, morepreferably from 0.05-0.25% by weight of the seed. Depending on the levelof expression in the seed only part or the total of the seeds normallyused in the brewing process can be replaced by transgenic seeds. Whenhigh levels of expression are reached it is also possible to add thetransgenic seeds of other plant genera not normally used in the brewingprocess without changing too much to the brewing mix.

[0075] In the case of malt, preferably part of the malt has received aspecial treatment, as compared to traditional malt, wherein the malt hasbeen heated during kilning to maximize production of color and flavourcompounds. The remaining enzyme activity is negligible after thistreatment.

[0076] Expression of enzymes in seed as is disclosed in the presentinvention provides the possibility to circumvent the addition ofexogenous microbial enzymes to the brewing process. The costs of theproduction of transgenic seeds, containing the enzymes are much lowerthan the costs of the production of enzymes by fermentative processes.Furthermore, transgenic seeds are more conveniently used, since theyprovide a stable and manageable storage form of enzymes and are easy tohandle. The cost reduction and the convenience of use which are coupledwith the use of transgenic seeds are especially relevant in the processof the present invention, because the need is circumvented to applyseveral different enzymes from several microbial fermentations in thebeer brewing process.

[0077] The course of the process of the present invention is much morepredictable than the course of a process using malt as a source ofenzymes, since the transgenic seeds contain no other enzyme activitiesat high levels than those expressed by way of the introduced genes. Inaddition, the course of the process of the present invention is muchmore predictable than the course of a process using microbial enzymes,since microbial enzyme preparations display undefined and varying levelsof side activities.

[0078] One of the areas in which it can be especially used is in Africancountries where malt importation is banned. The enzymes in that casecould be expressed in sorghum, which then could be added to the brewingprocess.

[0079] Next to the applications in the brewing of alcoholic beveragesalso other applications can be foreseen, in which MaltSeed can replacemalted grains.

[0080] Malted barley and/or malted wheat are used by millers tostandardize the diastasic power of flour. Also hemicellulases (such asxylanase) are added to the flour to improve the gas retention capacityof doughs made from the flour. The addition or use of enzymes in flourcan be replaced by using MaltSeed, which is very suitable since theenzymes can be added in the form of grains which anyhow are used.Identically also in the baking process enzymes can be added as areplacement for the malt which is present in many doughs. Enzymes whichare improving the baking process are xylanase,amylase,arabinofuranosidase, exopeptidase. Also the glucose oxidase fromAspergillus niger can be expressed in seeds for bakery purposes.

Example 1 Activity Measurement of endo-β-1,4-xylanase

[0081] Endoxylanase is obtained from a pure culture of Aspergillus nigerin a sterile tank and medium. The culture medium contains appropriatecarbon and nitrogen sources just as mineral salts. The fermentation iscarried out at a constant temperature between 30-40° C. and pH ismaintained within the range 3-5.

[0082] The activity of the enzyme is measured by hydrolysis of xylanfrom oat spelts suspended (35 g/l) in 1M glycine buffer pH 2.75. Theviscosity of this solution is determined by using a capillary viscometer(Ubbelhode type) at 47° C. The time dt needed for the upper meniscus ofthe liquid to fall down between two reference points is measured withintime T. The slope of the plot T versus 1/dt yields an apparent kineticconstant. 1 Lyx unit is the amount of enzyme needed to reach a value of1 min-1 for that kinetic constant.

Example 2 Activity Measurement of Exopeptidase

[0083] A production strain of Aspergillus sojae (DS 8351) is cultured.Exopeptidase activity is expressed as Leucine aminopeptidase units(Leu-A): 1 Leu-A is the amount of enzyme needed to produce 1 μmolp-nitroaniline per minute at pH 7.2 and 20° C. fromL-leucine-p-nitroaniline. The test is performed as follows: Leucineparanitroanilid (SIGMA) is dissolved in water at a concentration of 9mM. 1 ml of the solution is mixed with 1.5 ml 0.1 M phosphate buffer pH7.2. At t=0, 0.5 ml enzyme is introduced and left for reaction at 20° C.Fifteen minutes later, 1 ml 1N HCl is added to stop the reaction. Ablank is run with 1N HCl being introduced at t=0. Optical density isdetermined for the blank (OD_(blank)) and for the assay (ODassay) at 400nm. Activity is calculated as follows:$A = {{\frac{\left( {{OD}_{blank} - {OD}_{assay}} \right)}{9.8 \times 15} \times \frac{4}{0.5}\quad {Leu}} - {A/{ml}}}$

Example 3 Activity Measurement of Arabinofuranosidase

[0084] Isoenzyme A or isoenzyme B or arabinoxylanhydrolase have beenobtained from a culture of Aspergillus niger or Aspergillus nidulansstrains. Activity of isoenzymes A and B is measured by the hydrolysis ofp-nitrophenyl-alpha-L-arabinofuranoside. 1 ARF unit is the amount ofenzyme needed to liberate 1 μmol p-nitrophenol per minute under the testconditions described in Gunata Z. et al. (J. Agric. Food Chem. 38, 772,1989).

Example 4 Activity Measurement of Saccharifying Amylase

[0085] Saccharifying amylase is obtained from a pure culture ofPenicillium emersonii in a sterile tank and medium, which containsappropriate carbon and nitrogen source just as mineral salts. The tankis fed with maltodextrines 10-30 hours (preferably 24 h) after the startof the fermentation. Temperature is maintained in the range of 40-50° C.(preferably 45° C.) and pH is maintained in the range 4.5-5.5(preferably 5.0). The fermentation is stopped 40-55 h (preferably 48 h)after start.

[0086] Saccharifying amylase activity is measured according to theBETAMYL test, commercially available from MEGAZYME, Ireland. 1 BTU isthe amount of enzyme needed to produce 1 μmol p-nitrophenol at pH 6.2and 40° C. from Megazyme's commercial substrate.

Example 5 Preparation of Wort using Microbial Enzymes

[0087] A wort was prepared from crude barley grains, variety PLAISANT.Barley was ground with the EBC MIAG mill in order to reach filter presstype granulometry. 57 g of the obtained milled barley was suspended in300 ml warm water (50° C.) and containing:

[0088] 650 Lyx units endo-β-(1,4)-xylanase

[0089] 850 ARF units arabinofuranosidase

[0090] 18 mg B.A.T.S. (thermostable amylase)

[0091] 6 mg Brewers Protease (+) (endo-protease)

[0092] 1 mg Filtrase L3000 (+) (β-(1,3;1,4)-glucanase)

[0093] The temperature was maintained at 50° C. for 30 minutes and thenraised up to 63° C. (rate 1° C./min); the temperature was furthermaintained at 63° C. for 30 minutes and then raised up to 72° C. (rate1° C./min) and maintained at that temperature for 30 minutes. It wasfinally heated up to 76° C. (rate 1° C./min) and maintained at thattemperature for 5 minutes. Water was added to compensate for waterevaporation. The mash was then poured into a funnel containingSchleicher and Schull paper filter. From the density of the filteredwort, yield was determined as done in any brewery; also viscosity andfree amino acids (FAA) levels were determined according to standard EBCprocedures.

[0094] The yield was 71.5%, viscosity was 2.52 mPa.s and 66 mg/l of freeamino acids were measured.

Example 6

[0095] Comparison of Saccharifying Amylases

[0096] A wort was prepared from crude barley grains, variety PLAISANT.Barley was ground with the EBC MIAG mill in order to reach filter presstype granulometry. 57 g of the obtained milled barley was suspended in300 ml warm water (50° C.) and containing:

[0097] 650 Lyx units endo-β-(1,4)-xylanase

[0098] 850 ARF units arabinofuranosidase

[0099] 18 mg B.A.T.S. (thermostable amylase)

[0100] 6 mg Brewers Protease (+) (endo-protease)

[0101] 100 Leu-A units exopeptidase

[0102] 1 mg Filtrase L3000 (+) (β-(1,3;1,4)-glucanase)

[0103] According to Table 1 saccharifying enzymes were added to the brewmixture. TABLE 1 Doses of saccharifying enzymes Brew no. Saccharifyingenzyme 1 none  0 2 Brewers Fermex 510 FAU 3 Amylase from P. emersonii 10 BTU 4 Brewers Fermex + amylase 510 FAU + 10 BTU from P. emersonii

[0104] The temperature was maintained at 50° C. for 30 minutes and thenraised up to 63° C. (rate 1° C./min); the temperature was furthermaintained at 63° C. for 30 minutes and then raised up to 72° C. (rate1° C./min) and maintained at that temperature for 30 minutes. It wasfinally heated up to 76° C. (rate 1° C./min) and maintained at thattemperature for 5 minutes. Water was added to compensate for waterevaporation. The mash was then poured into a funnel containingSchleicher and Schull paper filter. From the density of the filteredwort, yield was determined as done in any brewery; also viscosity andfree amino acids (FAA) levels were determined according to standard EBCprocedures.

[0105] Results of the measured yield, viscosity and FAA given in Table 2show the effects of the saccharifying enzyme of Penicillium emersonii asa substitute of Brewers Fermex whereas no real synergism can be expectedfrom the use of both enzymes. Particularly surprising is the quitepositive effect of the amylase of Penicillium emersonii on FAA increaseand viscosity reduction. TABLE 2 Results Viscosity FAA (12 Brew no.Yield (%) (mPa.s) Plato) (mg/l) 1 71.2 3.12 116 2 74.6 2.73 114 3 78.21.99 153 4 79.6 1.99 152

Example 7 Construction of a Binary Vector Containing a Seed-specificExpression Cassette

[0106] An expression construct is constructed in such a way thatseed-specific expression is obtained, using sequences of Oryza sativa L.glutelin storage protein (Zheng et al., Plant Physiol (1995) 109;77-786). These sequences may be replaced by those from similarseed-specific genes to achieve the same goal as is the objective of thisinvention.

[0107] For the construction of the expression construct forseed-specific expression, the promoter and terminator sequences from theglutelin (Gt1) gene of Oryza sativa L. are synthesized using PCRtechnology with the genomic clone Gt1 (Okita et al., J. Biol. Chem. 264,12573-12581,1989) as a template. This gene shows seed-specificexpression and its coding and flanking sequences have been determined(EMBL, Genbank Nucleotide Sequence Database accession number D00584)).Two sets of oligonucleotides are synthesized. One to allow amplificationof a 2.4 Kb fragment containing the Gt1 5′ flanking region encoding asan XhoI/SphI fragment: 5′Gt1.1 5′ GCACAATTCTCGAGGAGACCG 3′ 5′Gt1.25′ ATGGATGGCATGCTGTTGTAG 3′

[0108] The other amplification of the 3′ flanking sequences as aBamHI/EcoRI fragment (725bp): 3′Gt1.3 5′ CCTCTTAAGGATCCAATGCGG 3′3′Gt1.4 5′ CTTATCTGAATTCGGAAGCTC 3′

[0109] The oligos are designed to contain suitable restriction sites attheir termini to allow direct assembly of the expression construct afterdigestion of the fragments with restriction enzymes. Genes for theenzymes in the mixture according to the invention can be obtained fromliterature for the endo-xylanase (Mol. Microbiol. 12, 479-490, 1994),for the arabinofuranosidase isoenzyme A and isoenzyme B (EP 0 506 190),for the amylase from Bacillus licheniformis (EP 0 449 376), for theprotease from Bacillus amyloliquefaciens (J. Bact. 159, 811-819) and forthe glucanase form Bacillus amyloliquefaciens (Gene 49, 177-187, 1986).The genes for the saccharifying amylase from Penicillium and for theexopeptidase from Aspergillus sojae can easily be elucidated for aperson skilled in the art from the pure enzyme obtainable from thecultures indicated in the description. The codon usage of the genesencoding the enzymes to be expressed in seeds is optimized forexpression in monocot seeds. In order to do this the complete gene ismade synthetically, a BspHI site is introduced at the ATG start codonand a BamHI site is introduced down-stream of the TAA stop codon bothfor cloning purposes.

[0110] The 2.4 kb PCR product containing the 5′ flanking region of Gt1is digested with XhoI/SphI and cloned in a vector pSL1180 linearizedwith XhoI/SphI. The resulting vector is linearized with SphI/BamHI andused as a vector in a three-way ligation with the syntheticenzyme-encoding gene and, optionally an oligonucleotide duplex codingfor a targeting signal. Targeting can be effectuated to the vacuole, tothe apoplast, to the amyloplast or (with e.g. a KDEL-retention signal)to the endoplasmatic reticulum.

[0111] From this vector a fragment is isolated, containing the fusionsof the Gt1 glutelin promoter, optional signal sequence and the syntheticgene. This fragment is cloned in a three-way ligation with the 725 bpPCR product containing the 3′ terminator sequence of Gt1 digested withBamHI/EcoRI into binary vector pMOG22 (in E. coli K-12 strain DH5-alpha,deposited at the Centraal Bureau voor Schimmelcultures on Jan. 29, 1990under accession number CBS 101.90).

Example 8 Construction of a Binary Vector Containing the Endo-xylanaseGene in the Seed-specific Expressing Cassette

[0112] The endoxylanase gene from Aspergillus niger is used to optimisefor codon usage in barley. The resulting DNA sequence is depicted in SEQID NO:1.

[0113] For the expression of the endoxylanase gene extracellulartargeting is accomplished by the oligonucleotide duplex PRS 15′       AACTTCCTCAAGAGCTTCCCCTTTTATGCCTTCCTTTGTTTTGGCCAATACTTTGTAGCTGTTACGCATGC    3′PRS 23′ GTACTTGAAGGAGTTCTCGAAGGGGAAAATACGGAAGGAAACAAAACCGGTTATGAAACATCGACAATGCGTACGGTACC5′

[0114] encoding the signal peptide of the tobacco PR-S protein and forthe three-way ligation the synthetic xylanase gene digested withBspHI/BamHI is used.

[0115] From this vector a 3.1 Kb XhoI/BamHI fragment is isolated,containing the fusions of the Gt1 glutelin promoter, PR-S signalsequence and the synthetic xylanase gene. This fragment is cloned in athree-way ligation with the 725 bp PCR product containing the 3′terminator sequence of Gt1 digested with BamHI/EcoRI into binary vectorpMOG22. The resulting vector is designated pMOG1265.

Example 9

[0116] Barley Transformation

[0117] The method used for transformation of immature embryos of Hordeumvulgare cv. Golden Promise using Agrobacterium tumefaciens is generallyas described in Tingay, S. et al., The Plant J. 11(6), 1369-1376, 1997.In short, the protocol is as follows:

[0118] Donor plants for starting material are grown in a phytotron at10-20° C. with a 16 hr light period at 10,000-30,000 lux and 50-95% RH.Immature seed are harvested 10-15 days after pollination and sterilizedin a bleach solution for 20-40 minutes. Immature embryos are excisedfrom the young caryopses and the embryonic axis is removed with ascalpel blade. The explants are placed scutellum-side up on callusinduction medium and incubated at 24° C. in the dark for a periodranging from 16 hours till 7 days.

[0119] Embryos are immersed in an Agrobacterium suspension,approximately 0.1-10×10⁹ bacteria per ml, in which the Agrobacteriumcontains the constructs comprising the DNA encoding for the enzyme ofchoice, for 5 to 20 minutes and then transferred to callus inductionmedium. Thereupon, embryos are incubated for 2 or 3 days at 24° C. inthe dark. After coculture, embryos are transferred to callus inductionmedium containing antibiotics to kill Agrobacteria, directly combinedwith a selective agent to start the selection process of transgeniccells. The selection process occurs for up to 8 weeks. Resistantembryogenic callus lines are transferred to regenration medium andincubated in increasing light intensity (500 to 3000 lux) with a 16 hrlight period at 24° C. Regenerating plantlets are transferred to hormonefree or high cytokinin containing callus induction medium with orwithout selective agent. After development of a root system, plantletsare transferred to soil and grown to maturity with self-pollination.

Example 10

[0120] Three complete pilot brewing trials were performed. Two testbrews were done with a severely reduced amount of malt (20% of the rawmaterial) and the control with a normal amount of malt (See Table 3).The two test brews differed in the milling and filtering technology (SeeTable 3). The brewing diagram for all brews is shown in FIG. 1. TABLE 3Raw material composition, filtering and milling Control Test brew 1 Testbrew 2 Pils malt 75% 20% 20% Maize grits 25% 25% 25% Unmalted barley —55% 55% Filter Lautertun Lautertun Meura 20001 Milling Cylinder CylinderHammer

[0121] From the control mixture 8% malt was used in the cereal cookertogether with the maize grits, the other 67% malt was added to theconversion vessel. For the test brews 8% unmalted barley was used in thecereal cooker together with the maize grits, the other 47% unmaltedbarley was added to the conversion vessel together with the malt. TABLE4 Enzymes codes and amounts added per 100 kg raw material Enzyme CerealConversion Code Enzyme cooker vessel 1 Endo-β(1,4)-xylanase 117,000 Lyx2 Arabinofuranosidase 112,500 ARF 3 B.A.T.S. 16.5 g 4 Brewers Fermex 75g 5 Brewers protease (+) 75 g 6 Exo-peptidase — 7 Filtrase L3000 (+) 23g

[0122] The enzymes 1, 2, 4, 5 and 7 were added to the conversion vessel,while enzyme 3 was added to the cereal cooker (see Table 4 for enzymecodes and amounts added).

[0123] Wort processing results (mashing and lautering) were similar forthe test brews and the control. The two test brews did perform similarlyin the brewhouse. The test brews gave roughly the same wort, showingthat the differences in filtering and milling were not essential. From ataste comparison of the test and control beers it was concluded that allthree beers had a quite similar profile. A stronger mouthfeel wasobserved for the test brews in comparison with the control. This may bedue to a higher dextrin level that was found in the analysis of thewort. The amino acid levels in the wort were, although acceptable, lowerin the test brews in comparison with the control. The amino acid levelscan be increased by addition of the exopeptidase (see example 11). Thetest brew beers were classified by the tasting panel as good pilsenerbeers, showing that the partial replacement of malt by the enzymemixture resulted in a beer that is comparable to a beer manufacturedwith an amount of malt that is commonly used in the brewing industry.

Example 11

[0124] Two complete pilot brewing trials were performed. The test brewwas done with a reduced amount of malt and the control with a normalamount of malt (See Table 5). Filtering was done on the Lautertun. Forthe brewing diagrams of test brew 1 and the control see FIG. 2. TABLE 5Raw material composition Control Test brew 1 Pils malt 75% 20% Maizegrits 25% 25% Unmalted barley — 55%

[0125] From the control mixture 8% malt was used in the cereal cookertogether with the maize grits, the other 67% malt was added to theconversion vessel. For the test brew 8% unmalted barley was used in thecereal cooker together with the maize grits, the other 47% unmaltedbarley was added to the conversion vessel together with the malt.

[0126] In the test brew, enzymes 1-7 were added to the conversionvessel, while enzyme 3 was added to the contents of the cereal cooker aswell (see Table 6 for enzyme codes and amounts of enzyme used.

[0127] Wort processing results (mashing and lautering) were similar forthe test brews and the control. The free amino acid nitrogen content inthe wort was similar for the test brew and the control. The test brewresulted in beer that by tasting as considered to have a quite similarprofile as the control. The test brew beer classified by the tastingpanel as good pilsener beer, showing that the partial replacement ofmalt by the enzyme mixture resulted in a beer that is comparable to abeer manufactured with an amount of malt that is common use in thebrewing industry. TABLE 6 Enzymes codes and amounts added per 100 kg rawmaterial in the test brew Enzyme Conversion Code Enzyme Cereal cookervessel 1 Endo-β(1,4)-xylanase 117,000 Lyx 2 Arabinofuranosidase 112,500AFR 3 B.A.T.S. 16.5 g 33.5 g 4 Brewers Fermex   75 g 5 Brewers protease(+)   75 g 6 Exo-peptidase 105,000 LeuA 7 Filtrase L3000 (+)   23 g

Example 12

[0128] Transgenic barley grains from seven lines, each expressing one ofthe enzymes shown in Table 9 are mixed in such amounts that theresulting MaltSeed, when mixed in as 10% of the crude barley, providesenzyme activities as shown in Table 9. The MaltSeed preparation is mixedand ground together with the non-transgenic barley, togetherconstituting 55% of the raw material in the test brew. The raw materialcompositions in the cereal cooker and the conversion vessel for the testbrew are shown in Table 8. For the control 8% malt was used in thecereal cooker together with the maize grits. The rest of the rawmaterial was used in the conversion vessel. The distribution of MaltSeedgives the enzyme activities as shown in Table 10 in the conversionvessel and the cereal cooker.

[0129] Two brewing trials were performed. The test brew was done with areduced amount of malt and the control with a normal amount of malt (SeeTable 7). For the test brew a higher sacharification temperature wasused (See Table 7). Filtering was done on the Lautertun. For the brewingdiagram of the control and test brew see FIG. 2. TABLE 7 Composition rawmaterial Control Test brew 1 Pils malt 75% 20% Maize grits 25% 25%Unmalted barley + MaltSeed — 55%

[0130] TABLE 8 Raw material composition per 100 kg total in the testbrew Cereal cooker Conversion vessel Raw material (kg) (kg) Malt 20Unmalted barley 7.2 42.3 MaltSeed 0.8 4.7 Maize grits 25 Total (kg) 3367

[0131] Wort processing results (mashing and lautering) were similar forthe test brew and the control. The test brew resulted in beers that bytasting was considered to have a similar profile as the control. Thetest brew was classified as a classical malt beer by tasting, showingthat malt can be (partially) replaced by enzymes provided throughtransgenic seeds expressing these. TABLE 9 Amount of enzyme activityadded through addition of MaltSeed (units per 100 kg raw material) intest brew 1 Enzyme Cereal Conversion Code Enzyme Cooker vessel 1Endo-β(1,4)-xylanase  19,915 Lyx 117,000 Lyx 2 Arabinofuranosidase 19,149 ARF 112,500 ARF 3 B.A.T.S. 107,250 TAU 630,094 TAU 4 BrewersFermex  57,872 FAU 340,000 FAU 5 Brewers protease (+) 115,745 PC 680,000PC 6 Exo-peptidase  17,872 Leu-A 105,000 Leu-A 7 Filtrase L3000 (+) 11,701 BGLU  68,745 BGLU

[0132]

1 8 557 base pairs nucleic acid double linear cDNA NO NO CDS 1..555/product= “mature protein” 1 ATG AGC GCG GGA ATC AAC TAC GTC CAG AAC TACAAT GGC AAC CTC GGC 48 Met Ser Ala Gly Ile Asn Tyr Val Gln Asn Tyr AsnGly Asn Leu Gly 1 5 10 15 GAC TTT ACT TAC GAC GAG TCA GCG GGA ACT TTCAGC ATG TAT TGG GAG 96 Asp Phe Thr Tyr Asp Glu Ser Ala Gly Thr Phe SerMet Tyr Trp Glu 20 25 30 GAT GGC GTG TCC TCA GAC TTC GTC GTG GGA CTG GGCTGG ACC ACT GGA 144 Asp Gly Val Ser Ser Asp Phe Val Val Gly Leu Gly TrpThr Thr Gly 35 40 45 TCA TCC AAT GCG ATC ACC TAC AGC GCC GAG TAC TCC GCGTCA GGA TCA 192 Ser Ser Asn Ala Ile Thr Tyr Ser Ala Glu Tyr Ser Ala SerGly Ser 50 55 60 GCC TCC TAT CTG GCC GTG TAC GGA TGG GTG AAC TAC CCG CAGGCC GAG 240 Ala Ser Tyr Leu Ala Val Tyr Gly Trp Val Asn Tyr Pro Gln AlaGlu 65 70 75 80 TAC TAC ATC GTG GAG GAT TAC GGA GAT TAC AAC CCA TGC AGCTCA GCG 288 Tyr Tyr Ile Val Glu Asp Tyr Gly Asp Tyr Asn Pro Cys Ser SerAla 85 90 95 ACC TCC CTC GGA ACT GTG TAC AGC GAC GGC TCC ACC TAC CAG GTCTGC 336 Thr Ser Leu Gly Thr Val Tyr Ser Asp Gly Ser Thr Tyr Gln Val Cys100 105 110 ACC GAC ACC CGC ACT AAC GAG CCG TCA ATC ACC GGC ACT TCC ACCTTC 384 Thr Asp Thr Arg Thr Asn Glu Pro Ser Ile Thr Gly Thr Ser Thr Phe115 120 125 ACC CAG TAC TTC AGC GTG CGC GAG TCC ACT CGC ACC TCA GGA ACCGTG 432 Thr Gln Tyr Phe Ser Val Arg Glu Ser Thr Arg Thr Ser Gly Thr Val130 135 140 ACC GTC GCG AAC CAC TTC AAC TTC TGG GCG CAG CAC GGA TTC GGCAAC 480 Thr Val Ala Asn His Phe Asn Phe Trp Ala Gln His Gly Phe Gly Asn145 150 155 160 AGC GAC TTT AAC TAC CAG GTG GTC GCA GTG GAG GCA TGG TCAGGA GCG 528 Ser Asp Phe Asn Tyr Gln Val Val Ala Val Glu Ala Trp Ser GlyAla 165 170 175 GGC TCA GCG TCC GTC ACT ATC AGC TCC TG 557 Gly Ser AlaSer Val Thr Ile Ser Ser 180 185 185 amino acids amino acid linearprotein 2 Met Ser Ala Gly Ile Asn Tyr Val Gln Asn Tyr Asn Gly Asn LeuGly 1 5 10 15 Asp Phe Thr Tyr Asp Glu Ser Ala Gly Thr Phe Ser Met TyrTrp Glu 20 25 30 Asp Gly Val Ser Ser Asp Phe Val Val Gly Leu Gly Trp ThrThr Gly 35 40 45 Ser Ser Asn Ala Ile Thr Tyr Ser Ala Glu Tyr Ser Ala SerGly Ser 50 55 60 Ala Ser Tyr Leu Ala Val Tyr Gly Trp Val Asn Tyr Pro GlnAla Glu 65 70 75 80 Tyr Tyr Ile Val Glu Asp Tyr Gly Asp Tyr Asn Pro CysSer Ser Ala 85 90 95 Thr Ser Leu Gly Thr Val Tyr Ser Asp Gly Ser Thr TyrGln Val Cys 100 105 110 Thr Asp Thr Arg Thr Asn Glu Pro Ser Ile Thr GlyThr Ser Thr Phe 115 120 125 Thr Gln Tyr Phe Ser Val Arg Glu Ser Thr ArgThr Ser Gly Thr Val 130 135 140 Thr Val Ala Asn His Phe Asn Phe Trp AlaGln His Gly Phe Gly Asn 145 150 155 160 Ser Asp Phe Asn Tyr Gln Val ValAla Val Glu Ala Trp Ser Gly Ala 165 170 175 Gly Ser Ala Ser Val Thr IleSer Ser 180 185 71 base pairs nucleic acid single linear cDNA NO NONicotiana tabacum 3 AACTTCCTCA AGAGCTTCCC CTTTTATGCC TTCCTTTGTTTTGGCCAATA CTTTGTAGCT 60 GTTACGCATG C 71 80 base pairs nucleic acidsingle linear cDNA NO YES 4 CCATGGCATG CGTAACAGCT ACAAAGTATT GGCCAAAACAAAGGAAGGCA TAAAAGGGGA 60 AGCTCTTGAG GAAGTTCATG 80 21 base pairs nucleicacid single linear cDNA NO NO 5 ATGGATGGCA TGCTGTTGTA G 21 21 base pairsnucleic acid single linear cDNA NO NO 6 GCACAATTCT CGAGGAGACC G 21 21base pairs nucleic acid single linear cDNA NO NO 7 CCTCTTAAGG ATCCAATGCGG 21 21 base pairs nucleic acid single linear cDNA NO NO 8 CTTATCTGAATTCGGAAGCT C 21

1. A process for the production of alcoholic beverages, to which amixture of enzymes is added, which mixture comprises at least anendo-β(1,4)-xylanase, an arabinofuranosidase, an alpha-amylase, anendo-protease and a β-(1,3;1,4)-glucanase.
 2. A process according toclaim 1, where the mixture also comprises a saccharifying amylase.
 3. Aprocess according to claim 1 or 2, where the mixture also comprises anexo-peptidase.
 4. A process according to any of claim 1-3, characterizedin that that alcoholic beverage is beer.
 5. A process according to anyone of the claims 1-4, characterized in that each of said enzymes isprovided by the seeds of an individual transgenic plant line.
 6. Aprocess according to any of claims 1-4, characterized in that that morethan one enzyme is provided by the seeds of an individual transgenicplant line.
 7. A process according to any of claims 1-6, characterizedin that at least one enzyme is provided by the seeds of an individualplant line.
 8. A process according to any of claims 5-7, characterizedin that the transgenic plant line is a barley plant line.
 9. Transgenicseeds for use in the production of alcoholic beverages, expressing theenzymes which are necessary in the process for production of saidbeverages.
 10. Transgenic seeds according to claim 9, expressing enzymesselected from the group of alpha-amylase, endo-β-1,4-xylanase,β-1,3-1,4-glucanase, endoprotease, arabinofuranosidase, saccharifyingamylase and exopeptidase.